CN105886592A - 一种基于食欲素受体1分子探针的药物活性成分筛选方法 - Google Patents
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Abstract
本发明涉及分子生物学领域，公开了一种基于食欲素受体1分子探针的药物活性成分筛选方法，本发明采用荧光共振能量转移的方法建立了食欲素受体1分子探针，该分子探针具有高灵敏度，低噪音，高通量的特点，不仅能筛选受体激动剂也能筛选拮抗剂/反向激动剂，并且筛选过程不收下游信号的干扰；经过合理设计的分子探针能够对中药混合物进行筛选，能一步到位的在受体水平上筛选到分子作用靶点明确的先导化合物。
Description
技术领域
本发明涉及分子生物学领域，特别是一种基于食欲素受体1分子探针的药物活性成分筛选方法。
背景技术
常规的细胞水平、动物水平的药物筛选方法不仅费时、费力，而且容易受到多重因素的干扰，假阳性、假阴性率较高，也很难确定药物作用的靶点，并且需要大量的被筛选化合物进十年来，随着生物检测技术和计算机技术的快速发展，分子水平的药物筛选方法不断涌现，如：表面等离子体共振法、核磁共振法、下游信号分子检测法、荧光偏振法、虚拟筛选法等等，每种方法有自己的优缺点，都属于间接的筛选方法。至今还没有以受体空间构象变化为指针，可以直接、实时监测受体对药物反应的高灵敏度的筛选法。
发明内容
本发明的目的在于提供一种基于食欲素受体1分子探针的药物活性成分筛选方法，根据受体空间构象变化为指针，可以直接、实时监测受体对药物反应的高灵敏度的筛选法。
为实现上述技术目的，达到上述技术效果，本发明公开了一种基于食欲素受体1分子探针的药物活性成分筛选方法，筛选方法包括了以下步骤：
步骤1：构建的食欲素受体1分子探针：
A.采用PCR的方法将VSV-G tag及限制性内切酶位点分别引入人食欲素的cDNA的5’端,并用Over lapping PCR法将eCFP的cDNA连接到受体cDNA的3’端，将“受体-eCFP”连接到pcDNA5/FRT/TO载体，从而得到pcDNA5/FRT/TO-VSV-受体-eCFP质粒；
B.用突变法将质粒中受体第三内环中部的12个氨基酸置换为F lAsH片段：FLNCCPGCCMEP，从而得原始的分子探针质粒；
C.用探针质粒转染HEK293细胞48小时,以表达受体分子探针，并用市售的受体激动剂激活探针，用高速荧光显微镜监测探针的灵敏度；
D.根据探针灵敏度的监测结果，对探针进行结构优化；
步骤2：建立诱导表达G蛋白偶联受体分子探针的Flp-In T-RexHEK293细胞系：
A.将优化后的分子探针质粒和pOG44载体共转染Flp-In T-RexHEK293细胞，并用200ug/ml Hygromycin筛选阳性细胞克隆；
B.用1ug/ml Doxycycline诱导细胞表达相应的G蛋白偶联受体分子探针，用VSV抗体检测探针蛋白的表达，用荧光显微镜监测分子探针的细胞定位；
C.用受体激动剂处理已经表达的受体分子探针Flp-InT-RexHEK293细胞，得到不同激动剂剂量的受体激活的FRET曲线，并在停止给药后继续用生理盐水洗脱激动剂。
其中，步骤1中对探针进行结构优化具体如下：
调整受体C末端非a螺旋区的长度：将受体C末端有65个氨基酸截短为50个氨基酸；优化前探针的eCFP位于受体氨基酸的425位置，优化后探针的eCFP位于受体氨基酸的410位置；调整FLNCCPGCCMEP片段在受体第三内环的位置：将受体第三内环截短，将FLNCCPGCCMEP片段置于受体氨基酸的252-286之间的位置。
本发明具有以下有益效果：
1.本发明采用荧光共振能量转移的方法建立了食欲素受体1分子探针，该分子探针具有高灵敏度，低噪音，高通量的特点，不仅能筛选受体激动剂也能筛选拮抗剂/反向激动剂，并且筛选过程不受下游信号的干扰。
2.经过合理设计的分子探针能够对中药混合物进行筛选，能一步到位的在受体水平上筛选到分子作用靶点明确的先导化合物。
附图说明
图1为本发明分子探针的结构示意图。
图2为本发明实施例2的结果示意图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。
实施例1
如图1所示，本发明公开了一种基于食欲素受体1分子探针的药物活性成分筛选方法，筛选方法包括了以下步骤：
步骤1：构建的食欲素受体1分子探针：
A.采用PCR的方法将VSV-G tag及限制性内切酶位点分别引入人食欲素的cDNA的5’端,并用Over lapping PCR法将eCFP的cDNA连接到受体cDNA的3’端，将“受体-eCFP”连接到pcDNA5/FRT/TO载体，从而得到pcDNA5/FRT/TO-VSV-受体-eCFP质粒；
B.用突变法将质粒中受体第三内环中部的12个氨基酸置换为FlAsH片段：FLNCCPGCCMEP，从而得原始的分子探针质粒；
C.用探针质粒转染HEK293细胞48小时,以表达受体分子探针，并用市售的受体激动剂激活探针，用高速荧光显微镜监测探针的灵敏度；
D.根据探针灵敏度的监测结果，对探针进行结构优化，调整受体C末端非a螺旋区的长度：将受体C末端有65个氨基酸截短为50个氨基酸；优化前探针的eCFP位于受体氨基酸的425位置，优化后探针的eCFP位于受体氨基酸的410位置；调整FLNCCPGCCMEP片段在受体第三内环的位置：将受体第三内环截短，将FLNCCPGCCMEP片段置于受体氨基酸的252-286之间的位置；
步骤2：建立诱导表达G蛋白偶联受体分子探针的Flp-InT-RexHEK293细胞系：
A.将优化后的分子探针质粒和pOG44载体共转染Flp-In T-RexHEK293细胞，并用200ug/ml Hygromycin筛选阳性细胞克隆；
B.用1ug/ml Doxycycline诱导细胞表达相应的G蛋白偶联受体分子探针，用VSV抗体检测探针蛋白的表达，用荧光显微镜监测分子探针的细胞定位；
C.用受体激动剂处理已经表达的受体分子探针Flp-InT-RexHEK293细胞，得到不同激动剂剂量的受体激活的FRET曲线，并在停止给药后继续用生理盐水洗脱激动剂。
实施例2
实验目的及方法：为了验证本发明制备分子探针的可行性和有效性，本实施例分别以生理盐水、阳性对照药物OXA和天麻粗提物分别研究三者对食欲素受体1分子探针荧光共振能量转移变化，具体的实验方法如实施例1所述，此处不再赘述。
实验结果：如图2所示，将表达食欲素受体1分子探针的细胞置于高速荧光显微镜下，分别用生理盐水、阳性对照药物和天麻粗提物处理细胞，监测食欲素受体1分子探针的荧光共振能量转移变化，实验证明食欲素受体1分子探针能对生理盐水并无明显反应、对阳性药物OXA发生高灵敏度的反应，对天麻粗提物也有与阳性药物OXA相似的反应，可以初步证明天麻粗提物含有激活食欲素受体1分子探针的活性成分。
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到的变化或替换，都应涵盖在本发明的保护范围之内。
Claims (2)
1.一种基于食欲素受体1分子探针的药物活性成分筛选方法，其特征在于，所述的筛选方法包括了以下步骤：
步骤1：构建的食欲素受体1分子探针：
A.采用PCR的方法将VSV-G tag及限制性内切酶位点分别引入人食欲素的cDNA的5’端,并用Over lapping PCR法将eCFP的cDNA连接到受体cDNA的3’端，将“受体-eCFP”连接到pcDNA5/FRT/TO载体，从而得到pcDNA5/FRT/TO-VSV-受体-eCFP质粒；
B.用突变法将质粒中受体第三内环中部的12个氨基酸置换为FlAsH片段：FLNCCPGCCMEP，从而得原始的分子探针质粒；
C.用探针质粒转染HEK293细胞48小时,以表达受体分子探针，并用市售的受体激动剂激活探针，用高速荧光显微镜监测探针的灵敏度；
D.根据探针灵敏度的监测结果，对探针进行结构优化；
步骤2：建立诱导表达G蛋白偶联受体分子探针的Flp-In T-RexHEK293细胞系：
A.将优化后的分子探针质粒和pOG44载体共转染Flp-In T-RexHEK293细胞，并用200ug/ml Hygromycin筛选阳性细胞克隆；
B.用1ug/ml Doxycycline诱导细胞表达相应的G蛋白偶联受体分子探针，用VSV抗体检测探针蛋白的表达，用荧光显微镜监测分子探针的细胞定位；
C.用受体激动剂处理的已经表达的受体分子探针Flp-InT-RexHEK293细胞，得到不同激动剂剂量的受体激活的FRET曲线，并在停止给药后继续用生理盐水洗脱激动剂。
2.如权利要求1所述的一种基于食欲素受体1分子探针的药物活性成分筛选方法，其特征在于：所述的步骤1中对探针进行结构优化具体如下：
调整受体C末端非a螺旋区的长度：将受体C末端有65个氨基酸截短为50个氨基酸；优化前探针的eCFP位于受体氨基酸的425位置，优化后探针的eCFP位于受体氨基酸的410位置；调整FLNCCPGCCMEP片段在受体第三内环的位置：将受体第三内环截短，将FLNCCPGCCMEP片段置于受体氨基酸的252-286之间的位置。
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| CN104321059A (zh) * | 2012-05-31 | 2015-01-28 | 默沙东公司 | 食欲素受体拮抗剂的固体剂量制剂 |
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| CN104321059A (zh) * | 2012-05-31 | 2015-01-28 | 默沙东公司 | 食欲素受体拮抗剂的固体剂量制剂 |
Non-Patent Citations (3)
| Title |
|---|
| MARTIN COTTET ET AL.: "BRET and time-resolved FRET strategy to study GPCR oligomerization:from cell lines toward native tissues.", 《FRONT ENDOCRINOL(LAUSANNE)》 * |
| TIAN-RUI XU ET AL.: "Intramolecular Fluorescence Resonance Energy Transfer(FRET) Sensors of the Orexin OX1 and OX2 Receptors Identify Slow Kinetics of Agonist Activation", 《THE JOURNAL OF BIOLOGICAL CHEMISTRY》 * |
| 崔志英等: "食欲素（Orexin）及其受体（OXR）的特点及功能", 《广东饲料》 * |
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